Transcription initiation defines kinetoplast RNA boundaries
AbstractMitochondrial genomes are often transcribed into polycistronic primary RNAs punctuated by tRNAs whose excision defines mature RNA boundaries. Although kinetoplast DNA lacks tRNA genes, it is commonly held that monophosphorylated 5′-ends of functional molecules typify precursor partitioning by an unknown endonuclease. To the contrary, we demonstrate that in Trypanosoma brucei individual mRNAs and rRNAs are independently synthesized as 3′ extended precursors. The transcription-defined 5′ terminus is converted into monophosphorylated state by the 5′ pyrophosphohydrolase complex, termed PPsome, which is activated by RNA editing substrate binding complex (RESC). Most guide RNAs lack PPsome recognition sites and, therefore, remain triphosphorylated. We provide evidence that both 5′ pyrophosphate removal and 3′ adenylation are essential for mRNA stabilization. Furthermore, we uncover a mechanism by which antisense RNA-controlled 3′-5′ exonucleolytic trimming defines mRNA 3′-end. We conclude that mitochondrial mRNAs and rRNAs are transcribed and processed as insulated units irrespective of their genomic location.SignificanceIt is commonly held that in trypanosomes both mitochondrial DNA strands are transcribed into polycistronic precursors. These primary RNAs are presumably partitioned into individual pre-mRNAs by a “cryptic” endonuclease. We challenged the polycistronic transcription/ endonuclease model after revealing precursor processing by 3′-5′ degradation. This work demonstrates individual transcription of each gene and mRNA 5′-end definition by the first incorporated nucleotide triphosphate. We have uncovered the stabilizing role of 5′ triphosphate to monophosphate conversion and identified a protein complex responsible for this reaction. We have discovered antisense noncoding RNA originating near mRNA 3′ end and showed that a duplex formation modulates exonuclease activity to delimit the mature 3′ end. Collectively, our findings reveal mechanisms by which transcription defines both mRNA termini.
